Compositions and method for relaxing smooth muscles

ABSTRACT

Compositions and methods for relaxing smooth muscle in a warm-blooded animal are provided, comprising the step of administering to the animal a transition-metal nitrosyl complex. In one aspect, the transition-metal nitrosyl complex is represented by the formula L 3  M(NO) y  X 3-y  where L is a two-electron Lewis base or L 3  is a six-electron Lewis base, M is a Group 6 or 8 transition-metal, and when y is 1, X is carbon monoxide, and when y is 2, X is a halide or pseudohalide. In another aspect, the transition-metal nitrosyl complex is represented by the formula  M(NO) 2  X y  ! 2  where X is a halide or pseudohalide, and when M is a Group 6 transition-metal, y is 2, and when M is a Group 8 transition-metal, y is 1. Methods are also described for treating hypertension, angina pectoris, congestive heart disease, and impotence utilizing pharmaceutical compositions comprising the above-described transition-metal nitrosyl complexes.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.08/132,632, filed Oct. 6, 1993, U.S. Pat. No. 5,631,284.

TECHNICAL FIELD

The present invention relates generally to compositions and methods ofrelaxing smooth muscles and, more specifically, to compositions andmethods which utilize transition-metal nitrosyl compounds as smoothmuscle relaxants.

BACKGROUND OF THE INVENTION

Muscle is divided into three types: skeletal, cardiac, and smooth.Briefly, smooth muscle is found in all muscles where contraction isinvoluntary, except for heart muscle. Generally, smooth muscle iscomposed of elongated, spindle-shaped, nucleated cells arranged parallelto one another and to the long axis of the muscle.

Smooth muscle is classified both functionally, and anatomically. Whenclassified functionally, it is broken into two groups: multi-unit, andsingle-unit smooth muscle. Briefly, multi-unit smooth muscle isactivated by nerves which cause contraction of independent muscle units,and thus is not spontaneously active. Examples of multi-unit muscle maybe found in large arteries and veins, the urinary bladder, and the irisand ciliary muscles of the eye. In contrast to multi-unit smooth muscle,single- unit smooth muscle is usually spontaneously active, and containstight junctions which facilitate the contraction of the muscle as asingle unit. Examples of single-unit smooth muscle may be found in thevasculature (small veins, small arteries, and arterioles), the bileduct, and the walls of the gastrointestinal and urinogenital systems(gut, ureter, and uterus).

When smooth muscle is anatomically classified, it is generally dividedinto two groups: vascular and non-vascular. Briefly, vascular smoothmuscle consists only of blood vessels (arteries, arterioles, and veins),whereas non-vascular muscle includes all other types of smooth muscle(e.g., gastrointestinal (stomach, duodenum, ileum, jejunum, caecum, andcolon), spleen, trachea/bronchus, seminal vesicle, ductus deferens,corpus cavernosum, biliary tract, ureter, and uterus).

Many diseases are characterized by the inability of smooth muscle torelax. For example, hypertension is a disease found in 15%-20% of alladults in the United States. This condition is characterized bypersistently high arterial blood pressure which, if left untreated, maylead to other serious cardiovascular diseases such as heart failure,ischemia, renal failure, and stroke. Hypertension is one of the mostimportant public health problems facing developed countries.

Because of the health risk associated with untreated hypertension, theimportance of treating those who suffer from elevated blood pressure hasbeen recognized. Clinical trials have shown that appropriatepharmacological treatment of patients with moderate or severehypertension reduces the risk of stroke, renal failure, and congestiveheart failure. Based upon these studies, patients are now commonlytreated with antihypertensive drugs in order to lower diastolic bloodpressure ("DBP") to levels below 90 mmHg. Common drugs which are usedfor this purpose include diuretics (hydrochlorothiazide,chlorthalidone), sympatholytics (clonidine, atenolol, prazosin),vasodilators (minoxidil, sodium nitroprusside), angiotensin convertingenzyme inhibitors (captopril, enalapril), and calcium entry blockers(nifedipine, nitrendipine). The selection of a specific drug dependsupon the severity of the disease, the patient's age and lifestyle, sideeffects, cost, and concomitant diseases and therapies.

One approach to the treatment of hypertension is to effect the reductionof elevated blood pressure through blood vessel dilation, which resultsin an increase of blood vessel diameter and concomitant blood pressurereduction. One class of compounds which are commonly utilized to dilateblood vessels are nitrovasodilators, which have been utilized for therelief of anginal attacks and the treatment of hypertension. Clinicallyuseful nitrovasodilators include organic nitrites and nitrates such asamyl nitrite, glyceryl trinitrate (nitroglycerin), isosorbide dinitrate,erythrityl tetranitrate, and pentaerythritol as well as an inorganicnitric oxide donor, sodium nitroprusside.

Nitroglycerin and sodium nitroprusside are the most commonly usednitrovasodilators. Nitroglycerin is therapeutically useful for thetreatment of angina pectoris and gastrointestinal spasm, while sodiumnitroprusside is primarily utilized for the treatment of hypertensiveemergencies. Nitroglycerin may either be administered at the time ofanginal attack or prophylactically in anticipation of exercise orstress. Although nitroglycerin is effective in relief of acute attack,continual exposure to nitroglycerin and other organic nitrates resultsin a reduction in the ability of these compounds to vasodilate bloodvessels. Because of the tolerance that develops from their chronic use,these nitrovasodilators have limited applicability.

Sodium nitroprusside is likewise effective in lowering blood pressure,although its use is primarily limited to hypertensive emergencies due toits efficacy and toxicity. In particular, the infusion of sodiumnitroprusside at high doses or for long periods of time (over 24 hours)is associated with the toxicities resulting from the accumulation ofcyanide and or thiocyanate.

Accordingly, there is a need in the art for physiologically acceptablesmooth muscle relaxants which do not result in the development oftolerance and do not have toxic side effects. The present inventionfulfills these needs, and further, provides other, related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides methods for relaxingsmooth muscle in a warm-blooded animal, comprising the step ofadministering to the animal a therapeutically effective amount of atransition-metal nitrosyl complex. Within one aspect of the invention,the transition-metal nitrosyl complex is represented by the formula L₃M(NO)_(y) X_(3-y) where L is a two-electron Lewis base or L₃ is asix-electron Lewis base, M is a Group 6 or 8 transition-metal, and wheny is 2, X is either a halide or pseudohalide, and when y is 1, X₂ iseither L₂ or XL.

Within another aspect of the invention, methods are provided forrelaxing smooth muscle in a warm-blooded animal, comprisingadministering to the animal a transition-metal nitrosyl complexrepresented by the formula M(NO)₂ X_(y) !₂ where M is a Group 6 or 8transition-metal, X is either a halide or pseudohalide, and when M is aGroup 6 transition-metal, y is 2, and when M is a Group 8transition-metal, y is 1.

A further aspect of the present invention provide methods for treatinghypertension in a warm-blooded animal, comprising administering to theanimal a therapeutically effective amount of a transition-metal nitrosylcomplex represented by either the formula L₃ M(NO)_(y) X_(3-y) or M(NO)₂X_(y) !₂ as described above.

Within another aspect of the present invention, methods for treatingangina pectoris in a warm-blooded animal are provided, comprisingadministering to the animal a therapeutically effective amount of atransition-metal nitrosyl complex represented by either the formula L₃M(NO)_(y) X_(3-y) or M(NO)₂ X_(y) !₂ as described above is disclosed.

A further aspect of the present invention provides methods for treatingcongestive heart failure in a warm-blooded animal, comprisingadministering to the animal a therapeutically effective amount of atransition-metal nitrosyl complex represented by either the formula L₃M(NO)_(y) X_(3-y) or M(NO)₂ X_(y) !₂ as described above.

Within a further aspect of the present invention, methods for treatingimpotence in a warm-blooded animal are provided, comprisingadministering to the animal a therapeutically effective amount of atransition-metal nitrosyl complex represented by either the formula L₃M(NO)_(y) X_(3-y) or M(NO)₂ X_(y) !₂ as described above is disclosed.

Another aspect of the present invention provides pharmaceuticalcompositions comprising a transition-metal nitrosyl complex and apharmaceutically acceptable carrier or diluent. The transition-metalnitrosyl complex of the pharmaceutical composition may be represented byeither the formula L₃ M(NO)_(y) X_(3-y) or M(NO)₂ X_(y) !₂ as describedabove. These pharmaceutical compositions may be utilized in the methodsdescribed above, as well as in a variety of in vitro and in vivo assayswhich are discussed in more detail below and in the Examples.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph which illustrates the effects of CpCr(NO)₂ Cl andCpMo(NO)₂ Cl on the relaxation of phenylephrine-preconstricted rataortic rings (1A) and the change in mean arterial blood pressure inconscious rats (1B).

FIG. 2 is a graph which illustrates the effects of pre-incubation withvehicle, CpCr(NO)₂ Cl, and nitroglycerin on subsequent relaxationresponses to CpCr(NO)₂ Cl, nitroglycerin, and sodium nitroprusside ofphenylephrine-preconstricted rat aortic rings.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is directed towardtransition-metal nitrosyl complexes which are effective in relaxingsmooth muscle in vivo, without the development of tolerance associatedwith organic nitrites and nitrates such as nitroglycerin, and withoutthe toxic side effects of sodium nitroprusside. As smooth musclerelaxants, these transition-metal nitrosyl complexes are useful aspharmaceutical compositions for the treatment of a variety of conditionswherein it is desired to relax smooth muscles, including for example,hypertension, angina pectoris, congestive heart failure, and impotence,as well as a variety of in vitro and in vivo assays described below.

The transition-metal nitrosyl complexes of the present invention may begenerally represented by the formula L₃ M(NO)_(y) X_(3-y) wherein L is atwo-electron Lewis base or L₃ is a six-electron Lewis base, wherein M isa transition-metal, wherein X is a halide or a pseudohalide, and whereinif y is 1, X is carbon monoxide and if y is 2, X is a halide orpseudohalide. As used herein, the term "transition-metal nitrosylcomplex" refers to a composition in which a transition-metal iscoordinatively complexed with ligands wherein at least one ligand isnitric oxide. The term "nitrosyl" refers to the nitric oxide ligand. Atransition-metal is defined as any metal that is a member of PeriodicGroups 3-12. Preferred transition-metals include Groups 6 and 8 metals;chromium, molybdenum, tungsten, and iron, ruthenium, osmium,respectively. A more preferred embodiment of the present inventioncomprises transition-metals from Group 6. As represented by the formulaL₃ M(NO)_(y) X_(3-y), the transition-metal ligands are L, NO (nitricoxide), and X wherein- L is a two-electron Lewis base or L₃ is asix-electron Lewis base and X is a halide or a pseudohalide, oralternatively, a two-electron Lewis base, L. As used herein, the term"Lewis base" refers to any chemical species which is an electron pairdonor. Two-electron Lewis bases are those bases which may donated asingle pair of electrons. Suitable two-electron Lewis bases includebases which bear atoms from Periodic Groups 15 and 16. Lewis bases fromGroup 15 contain nitrogen, phosphorous, arsenic, antimony or bismuthatoms as electron pair donors. Preferable Lewis base from Group 15contain nitrogen, phosphorous, and antimony, and more preferably,nitrogen or phosphorous. Nitrogen containing Lewis bases include ammoniaand its carbon substituted derivatives including primary, secondary, andtertiary amines and aromatic amines including pyridine and pyrrole aswell as saturated analogs, piperidine and pyrrolidine. Other preferablenitrogen containing Lewis bases include amino acids and theirderivatives. Phosphorous containing Lewis bases include phosphine andits carbon substituted derivatives including primary, secondary, andtertiary phosphines as well as alkoxy substituted derivatives includingprimary, secondary, and tertiary phosphites. Lewis bases from Group 16contain oxygen, sulfur, or selenium atoms as electron pair donors.Preferable Lewis bases from Group 16 contain oxygen or sulfur. Oxygencontaining Lewis bases include water and alcohols and sulfur containingbases include hydrogen sulfide and thiols. Carbon monoxide is also atwo-electron Lewis base. The transition-metal nitrosyl complex may alsohave a six-electron Lewis base as ligand. As represented by L₃ M(NO)_(y)X_(3-y), L₃ may represent a six-electron Lewis base. Six-electron Lewisbases include bases such cyclopentadienyl anion and derivatives. In apreferred embodiment, the six-electron Lewis base ispermethylcyclopentadienyl anion, and in a most preferred embodiment, thesix-electron Lewis base is cyclopentadienyl anion. The transition-metalnitrosyl complex may also bear ligand X wherein X may be carbon monoxidewhere y is 1, and wherein X may be either a halide or pseudohalide wherey is 2. Suitable halides include fluoride, chloride, bromide, andiodide. Preferred halides include chloride and iodide. Suitablepseudohalides include nitrite, nitrate, and cyanide anion.

In a preferred embodiment of the invention, the transition-metalnitrosyl complex may bear two nitrosyl ligands. In particular, thetransition-metal nitrosyl complex represented by the general formula L₃M(NO)_(y) X_(3-y) where y is 2 becomes L₃ M(NO)₂ X. In a most preferredembodiment, the six-electron Lewis base, L₃, is cyclopentadienyl anion;X may be selected from the group consisting of chlorides and nitro; andthe transition-metal, M, may be selected from the group consisting ofchromium, molybdenum, and tungsten.

In another preferred embodiment, the transition-metal may bear a singlenitrosyl ligand. The transition-metal nitrosyl complex represented bythe general formula L₃ M(NO)_(y) X_(3-y) where y is 1 becomes L₃M(NO)X₂. In a most preferred embodiment, the six-electron Lewis base,L₃, is cyclopentadienyl anion; X is carbon monoxide; and thetransition-metal, M, may be selected from the group consisting ofchromium, molybdenum, and tungsten.

In yet another embodiment, the transition-metal nitrosyl complex bears asingle nitrosyl ligand and is represented by the formula L₃ M(NO)X₂where X₂ may be XL. Such a transition-metal nitrosyl complex isrepresented by the formula L₃ M(NO)XL. In a most preferred embodimentthe six-electron Lewis, base, L₃, is cyclopentadienyl anion; X isiodide; the two-electron Lewis base, L, may be selected from the groupconsisting of pyridine, piperidine, and trimethylphosphite; and thetransition-metal, M, may be selected from the group consisting ofchromium, molybdenum, and tungsten.

Within another aspect of the present invention, transition-metalnitrosyl complexes of the present invention may be represented by theformula M(NO)₂ X_(y) !₂ wherein M is a Group 6 or 8 transition metal, Xis a halide or pseudohalide, and when M is a Group 6 transition-metal, yis 2, and when M is a Group 8 transition-metal, y is 1.

Some of the transition-metal nitrosyl complexes of the present inventionare soluble in water and may be formulated as solutions in normal saline(0.9% sodium chloride). Water-insoluble complexes or complexes whichhave difficulty dissolving in water may be dissolved in 100%dimethylsulfoxide (DMSO), 60% DMSO in water, or 1:1 DMSO:Tween 80followed by dilution with normal saline. Some complexes may require10-20 minutes sonication to effect solution.

The transition-metal nitrosyl complexes of the present invention may beformulated as pharmaceutical compositions suitable for administration toa warm- blooded animal. Such formulations may contain an effectiveamount of the transition- metal nitrosyl complex as well as one or morepharmaceutically acceptable carriers and diluents. More specifically,the pharmaceutical compositions of the present invention may beadministered as liquids, emulsions, or suspensions containing acceptablediluents such as alcohols and sterile water containing sugars, salts,preservatives, suspending agents, emulsifying agents, or administered aslotions, creams, or gels containing acceptable diluents, preservatives,or carriers to impart the desired texture, consistency, and viscosity.Such acceptable diluents and carriers are familiar to those skilled inthe art, and include (but are not limited to) emulsifying agents such asnon-ionic ethoxylated and non-ethoxylated surfactants, fatty alcohols,fatty acids, preserving agents, wax esters, triglyceride esters, steroidalcohols, phospholipids such as lecithin and cephalin, polyhydricalcohol esters, fatty acid esters, hydrophilic lanolin derivatives,hydrophobic beeswax derivatives, hydrocarbon oils (such as palm oil,coconut oil, and mineral oil), cocoa butter waxes, silicon oils, pHbalancers, and cellulose derivatives. The present invention may beadministered as tablets or capsules which may contain suitable diluents,or administered as inhalations, sprays or aerosols which may containpropellant, or aqueous or oily vehicles, or administered assuppositories which may contain cocoa butter, glycerinated gelatin orother suitable bases.

Preferably, pharmaceutical compositions of the present invention areformulated for the particular method of administration (e.g.,intravenous, topical, pulmonary, rectal, sublingual, intramuscular, ororal), and in a manner which is in accordance with accepted practices,such as disclosed in Remington's Pharmaceutical Sciences, Gennaro, Ed.,Mack Publishing Co., Easton, Pa. 1990 (which is incorporated herein byreference in its entirety). In addition, pharmaceutical compositions ofthe present invention may be placed within containers, along withpackaging material which indicates that the pharmaceutical compositionmay be utilized to relax smooth muscles.

As noted above, the present invention also provides methods for relaxingsmooth muscle in a warm-blooded animal (e.g., humans, chimps, cows,goats, pigs, dogs, cats, rats or mice) comprising the step ofadministering to the animal a therapeutically effective amount of atransition-metal nitrosyl complex as described above. Briefly, theefficacy (and therapeutically effective amount) of a particulartransition-metal nitrosyl complex may be readily determined by both invitro and in vivo techniques. See generally Blattner et al., Experimentson Isolated Smooth Muscle Preparations, Hugo Sachs Elektronik KG, 1980(incorporated herein by reference in its entirety). For example, withinone embodiment, a small animal such as a rat, rabbit, or guinea-pig issacrificed and exsanguinated. The required tissue (e.g., artery, vein,stomach, ileum, jejunum, caecum, colon, uterus, spleen, trachea, seminalvesicle, ductus deferens, or corpus cavernosum) is removed and placed inan oxygenated physiological solution (e.g., Krebs or Tyrode solution).This tissue is then cut such that one end is attached to the bottom of amuscle bath, and the other end to a force-displacement transducer (forexample, Grass FT-03-C, Quincy, Mass.) for isometric recording with aphysiological recorder (Grass, Model 79G). An appropriate pre-load (0.5to 10 grams, depending on the tissue) is then applied, and the tissueequilibrated in the bath for one hour prior to the start of the study.

The measurement of smooth muscle contractile response may then beperformed essentially as described by Pang and Sutter in Blood Vessels17:293-301, 1980 (incorporated herein by reference in its entirety).Briefly, a dose-response curve of an agonist (e.g., phenylephrine,norepinephrine) is first obtained by measuring contractile response ofthe muscle as a function of agonist concentration. Response to theagonist is then measured beginning with the addition of the lowest doseof agonist to the bath. Each dose is equilibrated in the bath for 2 to 6minutes in order to assure a steady state response, prior to proceedingto the next higher dose.

Measurement of the effectiveness of smooth muscle relaxant compositionsmay then be determined by measurement of the smooth muscle relaxationresponse, in a manner analogous to contractile response. See generallyWang, Poon and Pang, J. Pharm. Exp. Ther. 265:112-119, 1993(incorporated herein by reference in its entirety). Briefly, tissue isfirst contracted with an agonist such as phenylephrine, usually at aconcentration which results in 90% of the tissue's maximum contractileresponse (referred to EC₉₀). At the steady state phase of thecontractile response to the agonist, a cumulative dose-response curvefor the smooth muscle relaxant may be determined. For each concentrationof relaxant, a steady state is achieved and the relaxation responsemeasured. The effective concentration (EC) of a relaxant is theconcentration that produces relaxation of a contraction previouslyinduced by an agonist. For example, the EC₅₀ of a relaxant is theeffective concentration of the relaxant required to relax apre-contracted muscle to 50% of its maximum contractile response.

As described in more detail below, transition-metal nitrosyl complexesof the present invention have been demonstrated to be effective in therelaxation of smooth muscle in vitro. In particular, a series oftransition-metal nitrosyl complexes were tested for their ability torelax pre-constricted rat aortic rings in vitro. All of thetransition-metal nitrosyl complexes completely and dose-dependentlyrelaxed phenylephrine pre-constricted aortae with efficacies similar tothat of sodium nitroprusside. The results of the in vitro relaxationexperiments are presented in Example 5 and Table 1. (See Example 5 forexperimental detail.)

Likewise, in vivo tests may also be readily developed and utilized inorder to determine the efficacy (and therapeutically effective amount)of the transition-metal nitrosyl complexes described above. For example,within one aspect of the present invention, a series of transition-metalnitrosyl complexes were tested in vivo for their ability to reduce themean arterial blood pressure of conscious rats in vivo. Briefly, asdescribed in greater detail below in Example 6, conscious, unrestrainedrats were intravenous bolus injected with the transition-metal nitrosylcomplexes. Five of the complexes were determined to dose-dependentlyreduce mean arterial pressure with maximum depressor effects whichranged from -36 to -59 mm Hg. Maximum depressor effects of sodiumnitroprusside and nitroglycerin under similar conditions were -58 and-56 mm Hg, respectively. Three of the complexes exhibited potenciessimilar to, or greater than, that of nitroglycerin, and two of thecomplexes were found to be more potent than sodium nitroprusside. Theresults of the effects of the transition-metal nitrosyl complexes on thedepression of mean arterial pressure in conscious rats in vivo aresummarized in Example 5 and Table 1.

In addition to determining efficacy of transition-metal nitrosylcomplexes in either in vitro or in vivo tests, it is also generallypreferably to determine whether the composition induces tolerance. Forexample, within one embodiment of the invention, induction of toleranceby the transition-metal nitrosyl complex described above may be readilydetermined by exposing rat aortic rings to a transition-metal nitrosylcomplex prior to relaxation determinations. More specifically, asdescribed in greater detail 10 below in Example 7, intact rat aorticrings were pre-incubated with a typical transition- metal nitrosylcomplex for one hour prior to examination of itsconcentration-relaxation effect in phenylephrine-preconstrictedconditions. Unlike the relaxation response of nitroglycerin, therelaxation response of the transition-metal nitrosyl complexes was notdiminished by pre-exposure of the aortic ring to the complex. Theresults indicate that tolerance develops less rapidly with thetransition-metal nitrosyl complexes of the present invention than withnitroglycerin.

As noted above, transition-metal nitrosyl complexes of the presentinvention may be readily administered to a warm-blooded animal in orderto relax a variety of smooth muscles. For example, within one aspect ofthe present invention, therapeutically effective amounts of atransition-metal nitrosyl complex may be administered to a warm-bloodedanimal in order to relax vascular smooth muscles. Through such methods,transition-metal nitrosyl complexes may be utilized in order to treat avariety of vascular disorders, including for example, hypertension,angina pectoris, and congestive heart failure.

The present invention also provides methods for relaxing nonvascularsmooth muscle through the administration of the transition-metalnitrosyl complexes described above. For example, within one aspect ofthe present invention methods are provided for treating impotence,comprising the step of administering to a warm-blooded animal atransition-metal nitrosyl complex as described above. The effectivenessof the transition-metal nitrosyl complexes described above for thetreatment of impotence may readily be evaluated through both in vitroand in vivo investigation. More specifically, in vitro evaluation may beaccomplished utilizing corpus cavernosum excised from the penis of arabbit, which is then dissected free from the tunica albuginea, cut intostrips and mounted at resting tension. Phenylephrine-precontractedstrips may be used in procedures similar to those described above, andin the examples, in order to construct dose-response curves for thetransition-metal nitrosyl complexes.

Similarly, in vivo studies may likewise be utilized in order to test theefficacy of the transition-metal nitrosyl complexes described above inthe treatment of impotence. Briefly, previous in vivo studies involvingthe systemic or central administration of low doses of apomorphine andother dopaminergic agonists, have shown that penile erection and yawningcan be reliably induced in rats (Heaton et al., J. Urol. 145:1099-102,1991). Therefore, in vivo studies may be utilized in order toinvestigate the utility of the transition-metal nitrosyl complexes ofthe present invention to facilitate apomorphine-induced penile erectionin rats. For example, within one embodiment of the invention rats areindividually placed in Plexiglas cages which have a mirror arranged inan oblique position to enhance observation. Pretreatment of the ratswith a transition-metal nitrosyl complex may then be utilized togenerate dose-response curves for apomorphine-induced rat penileerections.

Within another aspect of the present invention, methods are provided forenhancing learning and memory in warm-blooded animals, comprising thestep of administering to a warm-blooded animal one of theabove-described transition-metal nitrosyl complexes. The efficacy ofsuch complexes in enhancing memory and learning may be readilyascertained through a variety of tests, including, for example, theMorris water maze performance test for rats. See Morris, Learn Motiv.12:239-60, 1981; Decker and Majchrzak, Psychopharmacol. 107:530-34,1992. Briefly, rats are given daily injections of various doses oftransition-metal nitrosyl complexes at a time just prior to swimmingsessions. The rats are then trained several times daily over a period ofseveral days in a pool filled with warm milk. In each training trial,the rats are given a short period to find a submerged escape platformplaced in one of the quadrants of the pool. Those not locating theplatform will be guided to it. The mean escape latency on each trainingday may be determined as an index of information acquisition. The amountof time the rat spends in the training quadrant where the escapeplatform had been located during training will be indicative of acquiredspatial information. The effect of transition-metal nitrosyl complexeson acquired spatial information may reflect the utility of thesecomplexes in learning and memory.

Transition-metal nitrosyl complexes of the present invention havedemonstrated beneficial vasodilation properties through their capacityto relax blood vessels in vitro and depress blood pressure in vivo.Furthermore, the complexes are as effective in vasodilation as existingnitrovasodilators, nitroglycerin and sodium nitroprusside. Additionally,the complexes do not appear to cause the adverse effect of toleranceassociated with nitroglycerin nor toxicity associated with eithernitroglycerin or sodium nitroprusside.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES

Examples 1-4 describe the preparation of the transition-metal nitrosylcomplexes. All manipulations of compounds described in Examples 14 wereperformed under an atmosphere of prepurified dinitrogen or argon. Allsolvents were distilled from appropriate drying agents and degassedprior to use. Standard Schlenk and glove-box techniques were usedthroughout. Unless otherwise indicated, all other reactions andmanipulations were carried out in a well ventilated fume hood. In theseexamples, the following abbreviations are used: THF, tetrahydrofuran;CH₂ Cl₂, dichloromethane; IR, infrared spectrum; v refers to frequency;EIMS, electron impact mass spectroscopy; P⁺ refers to parent ion; ¹ HNMR, proton nuclear magnetic resonance; CDCl₃, deuteriochloroform; mp,melting point; FAB, fast atom bombardment; P(OMe)₃, trimethylphosphite;C₅ H₁₁ N, pip, piperidine; C₅ H₅ N, py, pyridine.

Examples 5-7 present in vitro and in vivo results of the evaluation ofthe efficiency of the transition-metal nitrosyl complexes in relaxingblood vessels and lowering blood pressure. Sprague-Dawley rats(300-400g) were used for all experiments. Each group consisted of 1-6rats for the in vivo experiments and 3-6 aortic rings for the in vitroexperiments. Each aortic ring was from a different rat.

EXAMPLE 1 The Synthesis of Transition-Metal Nitrosyl Complexes withFormula L₃ M(NO)_(y) X₂

Example 1 presents the syntheses of the transition-metal nitrosylcomplexes generally represented by the formula L₃ M(NO)_(y) X_(3-y)where y is 1: L₃ M(NO)X₂.

A. Preparation of Dicarbonyl(η⁵ -cyclopentadienyl) nitrosyl Complexes ofChromium, Molybdenum, and Tungsten

Briefly, a 200-mL, three-necked flask is fitted with a nitrogen inletand stirrer and is thoroughly flushed with prepurified dinitrogen. A dryTHF solution (50 mL) containing 4.18 g of sodium cyclopentadienide, NaC₅ H₅ ! is syringed into the flask, and Cr(CO)₆ (11.00 g, 50.0 mmol) and100 mL of di-n-butyl ether are added. (In the case of molybdenum andtungsten the di-n-butyl ether is not added.) The flask is then equippedwith a Leibig condenser and the reaction mixture is refluxed withvigorous stirring for 12 h. During this time, the reaction vessel isshaken occasionally to reintroduce any sublimed Cr(CO)₆ into therefluxing reaction mixture. The final reaction mixture is allowed tocool to room temperature and filtered, and the pale-yellow solid thuscollected is washed with diethyl ether (3×10 mL), and dried undernitrogen. The excess Cr(CO)₆ and any di-n-butyl ether remaining in thissolid are removed by sublimation at 90° C./0.005 torr onto awater-cooled probe. The Na (η⁵ -C₅ H₅)M(CO)₃ ! complexes of molybdenumand tungsten are freed of any unreacted hexacarbonyl in a similarfashion, and all three sodium salts are used without furtherpurification.

The preparations of all three (η⁵ -C₅ H₅)M(CO)₂ (NO) complexes fromtheir corresponding (η⁵ -C₅ H₅)M(CO)₃ !- anions are similar. Theexperimental procedure using the tungsten complex dicarbonyl(η⁵-cyclopentadienyl)nitrosyltungsten, is given below.

A 300-mL, three-necked flask is equipped with a nitrogen inlet, anaddition funnel, and a stirrer. It is charged with Na (η⁵ -C₅ H₅)W(CO)₃! (17.3 g, 48.5 mmol) and THF (120 mL). A THF solution (50 mL)containing Diazald (N-methyl-N-nitroso-p-toluenesulfonamide, AldrichChemical Co.) (10.4 g, 48.6 mmol) is syringed into the addition funnel.The solution of Diazald is added dropwise over a period of 15 minutesand the solvent is removed in vacuo. Sublimation of the resulting brownresidue at 50°-60° C./0.005 torr onto a water-cooled probe for threedays affords (η⁵ -C₅ H₅)W(CO)₂ (NO) (13.6 g, 84% yield).

The corresponding chromium and molybdenum complexes are obtainedsimilarly in yields of 60 and 93% respectively. The compounds are orangeto orange-red solids readily soluble in organic solvents. The solids arestable in air for short periods of time and indefinitely underdinitrogen.

Analysis calculated for C₅ H₅ Cr(CO)₂ (NO): C, 41.39; H, 2.48; N, 6.90.Found: C, 41.40; H, 2.60; N, 6.70. IR (CH₂ Cl₂): v_(CO) 2020, 1945;v_(NO) 1680 cm⁻¹. EIMS (probe temperature 120° C.): m/z 203 P⁺ !.

Analysis calculated for C₅ H₅ Mo(CO)₂ (NO): C, 34.03; H, 2.04; N, 5.67.Found: C, 34.28; H, 2.24; N, 5.54. IR (CH₂ Cl₂): v_(CO) 2020, 1937;v_(NO) 1663 cm⁻¹. EIMS (probe temperature 120° C.): m/z 249 P⁺ !.

Analysis calculated for C₅ H₅ W(CO)₂ (NO): C, 25.10; H, 1.50; N, 4.18.Found: C, 25.29; H, 1.70; N, 4.13. IR (CH₂ Cl₂): v_(CO) 2010, 1925;v_(NO) 1655 cm⁻¹. EIMS (probe temperature 120° C.): m/z 319 P⁺ !.

B. Preparation of Dicarbonyl(η⁵ -permethylcyclopentadienyl)nitrosylComplexes of Molybdenum and Tungsten

The permethylated complexes, (η⁵ -C₅ Me₅)M(CO)₂ (NO) (M=Mo, W) areprepared in a manner similar to that of their cyclopentadienyl analoguesexcept that Li C₅ Me₅ ! is used instead of Na C₅ H₅ !, and that highertemperatures (100° C.) are required to effect their sublimation.

Analysis calculated for C₁₀ H₁₅ Mo(CO)₂ (NO): C, 36.17; H, 4.55; N,4.22. Found: C, 36.17; H, 4.60; N, 4.30. IR (CH₂ Cl₂): v_(NO) 1659 cm⁻¹.EIMS (probe temperature 120° C.): m/z 333 P⁺ !. ¹ H NMR (CDCl₃) d 1.95(s).

Analysis calculated for C₁₀ H₁₅ W(CO)₂ (NO): C, 22.93; H, 1.92; N, 5.34.Found: C, 22.96; H, 1.87; N, 5.26. IR (CH₂ Cl₂): v_(NO) 1690 cm⁻¹. EIMS(probe temperature 120° C.): m/z 421 P⁺ !.

EXAMPLE 2 The Synthesis of Transition-Metal Nitrosyl Complexes withFormula L₃ M(NO)₂ X

This example presents the synthesis of the transition-metal nitrosylcomplexes generally represented by the formula L₃ M(NO)_(y) X_(3-y)where y is 2: L₃ M(NO)₂ X.

A. Preparation of Chloro(η⁵ -cyclopentadienyl)dinitrosyl Complexes ofChromium, Molybdenum, and Tungsten

Approximately 2-3 mL of nitrosyl chloride is condensed into a 5-mLgraduated cold trap held at -78° C. It is then allowed to melt so thatits volume can be measured, and it is distilled under static vacuum intoa 100-mL, two-necked flask held at -78° C. until just prior to use whenit is allowed to warm to room temperature.

All three (η⁵ -C₅ H₅)M(NO)₂ Cl complexes are prepared in a similarmanner, but to achieve maximum yields of the chromium and molybdenumcompounds, the reactions should be performed at -78° C. The molybdenumcomplex (chloro(η⁵ -cyclopentadienyl)nitrosylmolybdenum), on the otherhand, can be obtained in excellent yields even at room temperature asdisclosed below.

A 200-mL, three-necked flask, equipped with a stirrer, a nitrogen inlet,and an addition funnel, is charged with (η⁵ -C₅ H₅)Mo(CO)₂ (NO) (6.5 g,26 mmol) and CH₂ Cl₂ (100 mL). The nitrosyl chloride solution (typicallycontaining 2.3 mL (50 mmol) of nitrosyl chloride in 30 mL of CH₂ Cl₂) isadded dropwise to the stirred reaction mixture. Gas evolution occurs andthe orange solution becomes dark green. The reaction is monitored byinfrared spectroscopy and the nitrosyl chloride solution is added untilthe carbonyl absorptions of the initial organometallic reactant havedisappeared. It is extremely important that a stoichiometric amount ofnitrosyl chloride (CINO) be used, since even a slight excess of CINOreduces significantly the yields of the desired products, especially inthe cases of the chromium and tungsten complexes. Conversely, if aninsufficient quantity of CINO is added, the separation of any unreacted(η⁵ -C₅ H₅)M(CO)₂ (NO) from the desired chloronitrosyl complex isdifficult. The final reaction mixture is concentrated in vacuo toapproximately 30 mL, and is filtered through a short (3×5 cm) Florisilcolumn. The column is washed with CH₂ Cl₂ until the washings arecolorless, and the combined filtrates are then concentrated in vacuo toa volume of 30 mL. Dry hexanes are added until crystallization appearsto be complete; approximately 100-125 mL of hexanes are required. Theresulting green crystals are collected by filtration, washed with hexane(2×15 mL) and dried under dinitrogen to obtain analytically pure (η⁵ -C₅H₅)Mo(NO)₂ Cl (6.0 g, 89% yield). The chromium and tungsten compoundsare obtained similarly (except that the reaction flask is maintained at-78° C. during the addition of the CINO) in yields of 77 and 72%,respectively.

Analysis calculated for C₅ H₅ Cr(NO)₂ Cl: C, 28.26; H,.2.37; N, 13.18;Cl, 16.68. Found: C, 28.2; H, 2.55; N, 12.86; Cl, 16.99. IR (CH₂ Cl₂):v_(NO) 1816, 1711 cm⁻¹. EIMS (probe temperature 120° C.): m/z 212 P⁺ !.mp 144° C. (dec).

Analysis calculated for C₅ H₅ Mo(NO)₂ Cl: C, 23.42; H, 1.96; N, 10.92.Found: C, 23.53; H, 1.90; N, 10.70. IR (CH₂ Cl₂): v_(NO) 1759, 1665cm⁻¹. EIMS (probe temperature 120° C.): m/z 258 P⁺ !. mp 116° C.

Analysis calculated for C₅ H₅ W(NO)₂ Cl: C, 17.44; H, 1.46; N, 8.13; Cl,10.29.. Found: C, 17.68; H, 1.68; N, 8.10; Cl, 10.26. IR (CH₂ Cl₂):v_(NO) 1733, 1650 cm⁻¹. EIMS (probe temperature 120° C.): m/z 343P^(+!). mp 127° C. (dec).

B. Preparation of Nitrito(η⁵ -cyclopentadienyl)dinitrosylchromium

CpCr(NO)₂ Cl (1.0 g, 4.7 mmol) (Cp=η⁵ -C₅ H₅) is dissolved in 50 mLdistilled water. A distilled water solution (25 mL) of AgNO₂ (0.72 g,4.7 mmol) is added to the stirred, green solution of CpCr(NO)₂ Cl. Aflocculent white solid forms immediately. Stirring is continued for 30min, and then the mixture is filtered to remove the white solid. Thefiltered solution is extracted with 3×50 mL of CH₂ Cl₂. The extracts arecombined, dried with anhydrous Na₂ SO₄, and filtered. The solvent isremoved in vacuo until the solution reaches a volume of 20 mL. Twenty mLof hexanes are then added, and the mixture is cooled to -30° C. toinduce crystallization. This procedure results in the formation ofanalytically pure CpCr(NO)₂ (ONO) (0.51 g, 49% yield).

Analysis calculated for C₅ H₅ Cr(NO)₂ (ONO): C, 26.92; H, 2.26; N,18.83. Found: C, 27.21; H, 2.15; N, 18.76. IR (CH₂ Cl₂): v_(NO) 1825,1719 cm⁻¹. ¹ H NMR (CDCl₃) d 5.78 (s). EIMS (probe temperature 120° C.):m/z 223 P⁺ !.

EXAMPLE 3

The Synthesis of Transition-Metal Nitrosyl Complexes with Formula L₃M(NO)XL

This example presents the syntheses of the transition-metal nitrosylcomplexes generally represented by the formula L₃ M(NO)_(y) X_(3-y)where y is 1 and X₂ is XL: L₃ M(NO)XL.

A. Preparation of Iodo(η⁵ -cyclopentadienyl)nitrosylchromium Dimer

To a stirred orange solution of CpCr(CO)₂ (NO) (2.03 g, 10.0 mmol) isadded solid I₂ (1.24 g, 4.90 mmol). Reaction occurs after 5 min asevidenced by gas evolution and a color change of the reaction mixture togreen-brown. After being stirred for 1 h to ensure completion of thereaction, the mixture is taken to dryness under reduced pressure.Crystallization of the residue from CH₂ Cl₂ /hexanes affords dark greenCpCr(NO)I!₂ (2.35 g, 88% yield)

Analysis calculated for C₅ H₅ Cr(NO)I!₂ : C, 21.92; H, 1.84; N, 5.11; I,46.33. Found: C, 22.00; H, 1.77; N, 5.00; I, 46.08. IR (CH₂ Cl₂): v_(NO)1673 cm⁻¹. EIMS (probe temperature 120 ° C.): m/z 548 P⁺ !. mp 119° C.(dec).

B. Preparation of Iodo(η⁵ -cyclopentadienyl)nitrosyl(L)chromium(L=P(OMe)₃, pip, py)

Olive green CpCr(NO)I!₂ (0.27 g, 0.50 mmol) is dissolved in CH₂ Cl₂ (20mL). To the stirred solution, 0.081 mL (1.0 mmol) of pyridine is addedby syringe. The color changes from dark green to a brighter green overthe period of 30 min. The solution is reduced in volume to approximately10 mL, and then filtered through alumina (1.5×3 cm) supported on amedium-porosity frit. The column is washed with CH₂ Cl₂ until thewashings are colorless. Hexanes (30 mL) are added, and the solution isreduced in volume until crystallization is initiated. The mixture isthen cooled to -30° C. overnight to complete crystallization. Thesolvent is removed by cannulation, and the dark green crystals arewashed with pentane (3×15 mL). This solid is dried in vacuo to obtain0.32 g (90% yield) of CpCr(NO)(py)I.

Analysis calculated for C₁₀ H₁₀ N₂ OCrI: C, 34.02; H, 2.85; N, 7.93.Found: C, 34.15; H, 2.82; N, 7.80. IR (Nujol mull): v_(NO) 1651 cm⁻¹.FAB mass spectrum: m/z 353 P⁺ !.

The preparation of CpCr(NO)(L)I where L=piperidine and L=P(OMe)₃ isanalogous to above where L=pyridine.

Data for CpCr(NO) P(OMe)₃ !I: 0.35 g, 88% yield. Analysis calculated forC₈ H₁₄ NO₄ PCrI: C, 24.13; H, 3.54; N, 3.52. Found: C, 24.19; H, 3.54;N, 3.49. IR (Nujol mull): v_(NO) 1650 cm⁻¹. FAB mass spectrum: m/z 398P⁺ !.

Data for CpCr(NO)(pip)I. Analysis calculated for C₁₀ H₁₆ N₂ OCrI: C,33.44; H, 4.49; N, 7.80. Found: C, 33.23; H, 4.46; N, 7.70. IR (Nujolmull): v_(NO) 1651 cm⁻¹. FAB mass spectrum: m/z 359 P⁺ !

EXAMPLE 4 The Synthesis of Transition-Metal Nitrosyl Complexes withFormula M(NO)_(y) X₂ !_(n)

This example presents the syntheses of transition-metal nitrosylcomplexes generally represented by the formula M(NO)₂ X_(y) !₂ when M isa Group 6 transition metal, y is 2, and when M is a Group 8 transitionmetal, y is 1.

A. Preparation of Dichloro Dinitrosyl Complexes of Molybdenum andTungsten

A 500-mL, three-necked round-bottom flask is equipped with a gas inletand an addition funnel, and is charged with powdered W(CO)₆ (17.6 g,50.0 mmol). The entire system is then thoroughly purged with prepurifieddinitrogen and CH₂ Cl₂ (90 mL) is added. The addition funnel is chargedwith a CH₂ Cl₂ solution of CINO (6 mL, 120 mmol). 6 mL of the CINOsolution is added to the flask, and a small amount of air (0.50 mL) isadded by syringe to initiate the reaction. The addition of the CINOsolution is carried out in a dropwise manner, the reaction beingmonitored by IR spectroscopy. When the IR band at 1977 cm⁻¹ due to theW(CO)₆ starting material has disappeared, the addition is halted, andthe solution is allowed to stir for a further 30 min. The greenprecipitate which has formed is collected on a glass frit, washed withCH₂ Cl₂ and dried in vacuo to obtain W(NO)₂ Cl)₂ !_(n) (14.52 g, 92%yield) as an olive green powder.

Analysis calculated for WN₂ O₂ Cl₂ : C, 0.00; H, 0.00; N, 8.90; Cl,22.52. Found: C, 0.23; H, 0.00; N, 8.88; Cl, 21.97. IR (Nujol mull):v_(NO) 1794, 1680 cm⁻¹.

The preparation of Mo(NO)₂ Cl₂ !_(n) is analogous to W(NO)₂ Cl)₂ !_(n)above. Analysis calculated for MoN₂ O₂ Cl₂ : N, 12.4; Cl, 31.3; Mo,42.3. Found: N, 12.7; Cl, 32.2; Mo, 41.1. IR (Nujol mull): v_(NO) 1805,1690 cm⁻¹. 91% yield based on MO(CO)₆.

B. Preparation of Iodo(dinitrosyl)iron Dimer

Dry acetone (250 mL) and 33.5 g (0.3 mol) of iron powder are added to a1000-mL three-necked round-bottom flask equipped with a gas inlet, a500-mL pressure-equalizing addition funnel equipped with a bubbler, anda mechanical stirrer. A solution containing 38.1 g (0.1 mol) of I₂ inEt₂ O (300 mL) is then added through the addition funnel dropwise over aperiod of 45-74 min. After the addition is complete, NO gas isintroduced into the flask at a rate sufficient to maintain a flow of gasof about one bubble per second through the bubbler. Stirring must besufficiently vigorous to ensure efficient uptake of the NO gas. The endof the reaction is marked by the cessation of the absorption of nitricoxide as determined by the use of the bubbler.

The solvent is removed in vacuo. When the black residue is completelydry, the desired product is sublimed onto a water-cooled probe. Theflask should be heated slowly (2° C./min) to 90° C. Explosivedecomposition can take place if the heating rate is excessive. Theproduct can be scraped off the probe in an inert atmosphere drybox andstored under dinitrogen.

Analysis calculated for Fe(NO)₂ I!₂ : Fe, 23.0; I, 52.3. Found: Fe,23.4; I 51.8. IR (Nujol mull): v_(NO) 1807, 1766 cm⁻¹.

EXAMPLE 5 Relaxation of Blood Vessels Utilizing Transition-MetalNitrosyl Complexes

Example 5 presents the results of the evaluation of the efficacy of thetransition metal nitrosyl complexes in blood vessel relaxation. In thesein vitro experiments, phenylephrine pre-constricted rat aortae weresubjected to increasing doses of the transition-metal nitrosylcomplexes, and the relaxation of the rat aortae measured.Concentration-response (relaxation) curves were prepared, and theability of the transition-metal nitrosyl complexes to relax the bloodvessels were compared and related to nitroglycerin and sodiumnitroprusside. The results of these experiments are reported in terms ofEC₅₀ (effective concentration to produce a 50% relaxation response) andE_(max) (percent relaxation of phenylephrine-induced contraction) andare tabulated in Table 1.

Briefly, rats were sacrificed by a blow on the head followed byexsanguination. The thoracic aorta was removed and cleared of connectivetissues. Four ring segments of 0.5 cm length each were prepared from oneaorta and suspended in separate organ baths. Each ring was connected toa Grass FT-03-C force-displacement transducer (Quincy, Mass.) forisometric recording with a pre-load of 1 g and was equilibrated for 1 hr(with 3 washouts) in normal Krebs solution (pH 7.4) at 37° C. with a gasmixture of 95% O₂ and 5% CO₂. The Krebs solution had the followingcomposition (10⁻³ M): NaCl, 118; glucose, 11; KCl, 4.7; CaCl₂, 2.5;NaHCO₃, 25; KH₂ PO₄, 1.2; MgCl₂ 6H₂ O, 1.2.

Phenylephrine (10⁻⁶ M, EC₉₀) was added to the baths and at 15-20 minlater, at the steady state phase of the contractile response tophenylephrine, a cumulative concentration-response curve of sodiumnitroprusside or a metal nitrosyl complex was constructed usingsequential doubling of the drug concentration. Each concentration of adrug was left in the bath for 2-4 min to ensure a plateau response isreached. The time taken to complete each concentration-response curvewas approximately 20 min. FIG. 1A shows concentration-response curves ofCpCr(NO)₂ Cl and CpMo(NO)₂ Cl.

                                      TABLE 1                                     __________________________________________________________________________    The effects of metal nitrosyl complexes on relaxation of pre-constricted      aortic rings in vitro and depression of mean arterial pressure in             conscious rats in vivo                                                                    In vitro                                                                              In vivo                                                                 EC.sub.50                                                                        E.sub.max                                                                          ED.sub.50                                                                         E.sub.max                                                                          Toxic dose                                     Entry                                                                            Name     n (μM)                                                                          (%)                                                                              n (μg/kg)                                                                        (mmHg)                                                                             (mg/kg)                                        __________________________________________________________________________    1  Na nitroprusside                                                                       6 0.052                                                                            -128                                                                             6 8.4 -58  10,000.sup.a                                   2  Nitroglycerin                                                                          6 0.30                                                                             -104                                                                             6 200 -56  --                                             3  CpCr(NO).sub.2 Cl                                                                      6 0.11                                                                             -118                                                                             6 4.4 -42  2,000.sup.a                                    4  CpMo(NO).sub.2 Cl                                                                      6 17 -113                                                                             6 400 -59  25,600.sup.b                                   5  CpW(NO).sub.2 Cl                                                                       6 2.0                                                                              -122                                                                             2 2976                                                                              -36  40,000.sup.c                                   6  CpCr(NO)(pip)I                                                                         5 0.45                                                                             -110                                                                             --                                                                              --  --   --                                             7  CpW(NO)(CO).sub.2                                                                      3 2.5                                                                              -107                                                                             3 --  biphasic.sup.d                                                                     500.sup.a                                      8   Mo(NO).sub.2 Cl.sub.2 !.sub.n                                                         6 81 -119                                                                             2 >5120                                                                             no effect                                                                          --                                             9   W(NO).sub.2 Cl.sub.2 !.sub.n                                                          6 335                                                                              -108                                                                             1 >5120                                                                             no effect                                                                          --                                             10  Fe(NO).sub.2 I!2                                                                      6 20 -108                                                                             2 554 -51  5120.sup.c                                     11 CpCr(NO).sub.2 (ONO)                                                                   6 0.1                                                                              -98                                                                              2 1.5 -45  1280.sup.a                                     __________________________________________________________________________     .sup.a denotes dose which caused convulsion                                   .sup.b denotes dose which caused death                                        .sup.c denotes dose which caused neither convulsion nor death.                .sup.d denotes depressor followed by pressor response.                   

EXAMPLE 6 Depression of Arterial Blood Pressure UtilizingTransition-Metal Nitrosyl Complexes

Example 6 describes the results of the determination of the efficacy ofthe transition-metal nitrosyl complexes in reducing blood pressure ofconscious, unrestrained rats. In these in vivo experiments, thedepression of rat arterial pressure was measured as a function oftransition-metal nitrosyl complex dose. Dose-response (pressuredepression) curves were constructed and the capacities of thetransition-metal nitrosyl complexes to reduce arterial pressure werecompared and related to nitroglycerin and sodium nitroprusside. Theresults of these experiments are reported in terms of ED₅₀ (effectivedose to produce a 50% pressure depression response) and E_(max) (maximumdecrease in blood pressure) and are summarized in Example 5, Table 1.

The rats were anaesthetized with halothane (4% in air for induction and1.5% in air for surgery). A polyethylene cannula (PE50) was insertedinto the left iliac artery for the measurement of mean arterial pressure(MAP) by a pressure transducer (P23DB, Gould Statham, Calif.). A PE50cannula was also inserted into the iliac vein for the administration ofthe vehicle or a drug. The rats were then put into a small cage andgiven>6 hr to recover from the effects of anaesthesia and surgery beforeuse. The rats were allowed to move freely during the recovery phase aswell as during the conduction of the experiments as described in Wangand Pang, Br. J. Pharmacol. 103:2004-8, 1991. Only one dose-responsecurve of a metal nitrosyl complex, sodium nitroprusside, nitroglycerinor vehicle (0.9% NaCl solution or dimethylsulfoxide solution) wasconstructed in each rat. All drugs or vehicle were intravenous bolusinjected at dose-intervals of 3-7 min, to allow the recovery ofdepressor responses, before the injection of the next higher dose.

Five of the eight metal nitrosyl complexes tested were found to lowerblood pressure in a dose-related manner. The maximum depressor responsesof some of the metal nitrosyl complexes were similar to those ofnitroglycerin and sodium nitroprusside. The potencies (inverse of ED₅₀)of some of the metal nitrosyl complexes were similar to or greater thanthose of nitroglycerin and sodium nitroprusside. FIG. 1B showsconcentration-response curves of CpCr(NO)₂ Cl and CpMo(NO)₂ Cl.

EXAMPLE 7

Example 7 presents the results of pre-exposing the rat aortae to thetransition-metal nitrosyl complexes to evaluate the development oftolerance. In these experiments, the rat aortic rings were pretreatedwith a transition-metal nitrosyl complex prior to the measurement ofdose-dependent relaxation as described above in Example 5.

In this study, the vehicles, CpCr(NO)₂ Cl (10⁻⁶ M), or nitroglycerin(3×10⁻⁴ M) was added to the baths for 1 h, followed by the washout ofdrug or vehicle, preconstriction with phenylephrine and the constructionof a concentration-relaxation curve of CpCr(NO)₂ Cl.

The results show that the pre-incubation with CpCr(NO)₂ Cl did not alterthe EC₅₀ or E_(max) of the compound (see FIG. 2A), suggesting thattolerance to the tested drug does not readily develop. Pre-incubationwith nitroglycerin shifted the nitroglycerin dose-response curve to theright with EC₅₀ significantly increased and E_(max) unaltered. See FIG.2B. This shows that tolerance develops to nitroglycerin. Pre-exposure tonitroglycerin did not alter the EC₅₀ or E_(max) of CpCr(NO)₂ Cl, showingthat no cross-tolerance exists between nitroglycerin and CpCr(NO)₂ Cl.See FIG. 2C. Pre-exposure to nitroglycerin slightly, but statisticallysignificantly (p<0.05), increased the EC₅₀ of sodium nitroprusside,suggesting a small degree of cross-tolerance between these two drugs.See FIG. 2D.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except by the appended claims.

We claim:
 1. A method for relaxing smooth muscle in a warm-bloodedanimal, comprising administering to said animal a therapeuticallyeffective amount of a transition-metal nitrosyl complex represented bythe formulaL₃ M(NO)_(y) X_(3-y) wherein L is a two-electron Lewis baseor L₃ is a six-electron Lewis base, M is a Group 6 or 8transition-metal, wherein y is 1 or 2, X is either a halide orpseudohalide, and when y is 1, X₂ is either L₂ or XL.
 2. A method fortreating hypertension in a warm-blooded animal, comprising administeringto said animal a therapeutically effective amount of a transition-metalnitrosyl complex represented by the formulaL₃ M(NO)_(y) X_(3-y) whereinL is a two-electron Lewis base or L₃ is a six-electron Lewis base, M isa Group 6 or 8 transition-metal, wherein v is I or
 2. and when y is 2, Xis either a halide or pseudohalide, and when y is 1, X₂ is either L₂ orXL.
 3. A method for treating angina pectoris in a warm-blooded animal,comprising administering to said animal a therapeutically effectiveamount of a transition-metal nitrosyl complex represented by theformulaL₃ M(NO)_(y) X_(3-y) wherein L is a two-electron Lewis base or L₃is a six-electron Lewis base, M is a Group 6 or 8 transition-metal,wherein v is 1 or 2, X is either a halide or pseudohalide, and when y is1, X₂ is either L₂ or XL.
 4. A method for treating congestive heartfailure in a warm-blooded animal, comprising administering to saidanimal a therapeutically effective amount of a transition-metal nitrosylcomplex represented by the formulaL₃ M(NO)_(y) X_(3-y) wherein L is atwo-electron Lewis base or L₃ is a six-electron Lewis base, M is a Group6 or 8 transition-metal, wherein v is 1 or 2, X is either a halide orpseudohalide, and when y is 1, X₂ is either L₂ or XL.
 5. A method fortreating impotence in a warm-blooded animal, comprising administering tosaid animal a therapeutically effective amount of a transition-metalnitrosyl complex represented by the formulaL₃ M(NO)_(y) X_(3-y) whereinL is a two-electron Lewis base or L₃ is a six-electron Lewis base, M isa Group 6 or 8 transition-metal, wherein y is 1 or 2 and when y is 2, Xis either a halide or pseudohalide, and when y is 1, X₂ is either L₂ orXL.
 6. A method for relaxing smooth muscle in a warm-blooded animal,comprising administering to said animal a therapeutically effectiveamount of a transition-metal nitrosyl complex represented by theformulaM(NO)₂ X_(y) !₂ wherein M is a Group 6 or 8 transition-metal, Xis either a halide or pseudohalide, and when M is a Group 6transition-metal, y is 2, and when M is a Group 8 transition-metal, yis
 1. 7. A method for treating hypertension in a warm-blooded animal,comprising administering to said animal a therapeutically effectiveamount of a transition-metal nitrosyl complex represented by the formulaM(NO)₂ X_(y) !₂ wherein M is a Group 6 or 8 transition-metal, X iseither a halide or pseudohalide, and when M is a Group 6transition-metal, y is 2, and when M is a Group 8 transition-metal, yis
 1. 8. A method for treating angina pectoris in a warm-blooded animal,comprising administering to said animal a therapeutically effectiveamount of a transition-metal nitrosyl complex represented by the formulaM(NO)₂ X_(y) !₂ wherein M is a Group 6 or 8 transition-metal, X iseither a halide or pseudohalide, and when M is a Group 6transition-metal, y is 2, and when M is a Group 8 transition-metal, yis
 1. 9. A method for treating congestive heart failure in awarm-blooded animal, comprising administering to said animal atherapeutically effective amount of a transition-metal nitrosyl complexrepresented by the formula M(NO)₂ X_(y) !₂ wherein M is a Group 6 or 8transition-metal, X is either a halide or pseudohalide, and when M is aGroup 6 transition-metal, y is 2, and when M is a Group 8transition-metal, y is
 1. 10. A method for treating impotence in awarm-blooded animal, comprising administering to said animal atherapeutically effective amount of a transition-metal nitrosyl complexrepresented by the formulaM(NO)₂ X_(y) !₂ wherein M is a Group 6 or 8transition-metal, X is either a halide or pseudohalide, and when M is aGroup 6 transition-metal, y is 2, and when M is a Group 8transition-metal, y is
 1. 11. A pharmaceutical composition, comprising atransition-metal nitrosyl complex represented by the formula M(NO)₂X_(y) !₂ wherein M is a Group 6 or 8 transition-metal, X is either ahalide or pseudohalide, and when M is a Group 6 transition-metal, y is2, and when M is a Group 8 transition-metal, y is 1, and apharmaceutically acceptable carrier or diluent.
 12. The pharmaceuticalcomposition of claim 11 wherein X is a halide selected from the groupconsisting of fluoride, chloride, bromide and iodide.
 13. Thepharmaceutical composition of claim 11 wherein X is a pseudohalideselected from the group consisting of nitrite, nitrate, and cyanideanion.
 14. The pharmaceutical composition of claim 11 wherein thetransition-metal nitrosyl complex is iodo(dinitrosyl)iron dimer.